Previous studies have concluded that activation of PDE3 in adipocytes, hepatocytes, pancreatic Beta cells, and xenopus oocytes, is important in effects of insulin on inhibition of lipolysis and glycogenolysis, and of IGF-1 on inhibition of insulin secretion and stimulation of oocyte maturation. Incubation of 3T3-L1 adipocytes with insulin results in tyrosine phosphorylation of IRS-1, and activation of IRS-1-associated PI3-K. During chromatography on AcA 34, a portion of the intracellular PKB pool, found in association with intracellular membranes, co-elutes and co-immunoprecipitates (co-ip) with membrane-associated M3B. In addition, insulin induces formation of large macromolecular complexes, identified during gel filtration chromatography (Superose 6) of solubilized 3T3-L1 adipocyte membranes, that apparently contain phosphorylated/activated M3B and various signaling molecules possibly involved in its activation by insulin, i.e. IRS-1, PI3K p85, PKB, HSP-90, 14-3-3. Confocal microscopy also indicated colocalization of PDE3B and PKB. Inhibition of PI3-K with wortmannin inhibits insulin-induced assembly of macromolecular complexes, and insulin-induced phosphorylation/activation of PKB and M3B, as well as their interaction and co-ip. Wild type recombinant M3B (rM3B) co-ip much more efficiently with recombinant p-PKB (phosphorylated/activated PKB) than dephospho PKB (PKB) or p-delta PKB (p-PKB lacking its PH domain). Interactions between rM3B and p-PKB, not PKB, were also observed during gel filtration chromatography, and were inhibited by M3B peptide containing phosphorylated S273 (p-S273), but not with S273 peptide. After incubation of truncated mutants of rM3B (lacking the N-terminal 196, 302 and 604 aa, respectively) with p-PKB, little or no M3B-604 or M3B-302 co-ip with p-PKB; M3B-196 co-ip with p-PKB, but to a lesser degree than intact M3B. Taken together, these and other studies suggest that insulin induces the assembly of macromolecular complexes involved in activation of M3B, and that the structural determinants for its interaction with PKB seem to reside in, or are regulated by, the N-terminal portion of M3B and PH domain of p-PKB To examine the role(s) of PDE3 isoforms, mice with targeted disruptions of PDE3A and PDE3B genes have been generated. Both isoproterenol and CL 316,243 increased serum glycerol and FFA to a greater extent in intact PDE3B KO than in WT mice. CL also stimulated lipolysis to a greater extent in adipocytes isolated from KO mice. After mice were fasted for 20 hrs, however, CL stimulated lipolysis to a greater extent in adipocytes from WT than from KO mice, consistent with the idea that KO adipocytes are in a more "active" state. Serum glycerol and FFA were significantly increased in PDE3B KO mice after 20 hrs fasting. Insulin inhibited lipolysis in adipocytes from fasting and non-fasting WT, but not KO, mice. Insulin-induced lipogenesis was increased in isolated KO adipocytes. In isolated pancreatic islets, incubation with 16.7 mM glucose, in the absence or presence of 100 nM GLP-1, increased insulin secretion to a greater extent in islets from KO mice from WT mice. Although, in intact mice, IP or IV administration of CL produced a greater increase in serum insulin in KO than in WT mice, reduction of blood glucose was not more pronounced in KO mice. Insulin tolerance tests and hyperinsulinemic-euglycemic clamps also indicated the presence of insulin resistance in KO mice, most likely at the level of the liver, since insulin did not inhibit endogenous glucose production in the KO mice. Insulin-induced glucose uptake was similar in adipocytes from WT and KO mice, consistent with clamp findings in intact mice and also suggesting that adipocytes are not the origin of insulin resistance. Liver triglyceride content was increased in KO mice; FAS contents were increased in both liver and adipocytes from KO mice. After fasting for six hrs, hepatic cAMP content as well as phosphorylation of PKA substrates and CREB, and PGC-1 and gluconeogenic gene expression were increased to a greater extent in KO livers. Increased expression of TRB-3 protein was observed, which might contribute to induction of insulin resistance in KO liver. Expression of several inflammation-related genes was also increased in 6-hr fasted KO livers. In addition, some insulin-signaling molecular components were compromised in KO livers. In conclusion, our studies with PDE3B KO mice confirm previously suggested roles for PDE3B in the mediating the antilipolytic action of insulin and effects of cAMP on insulin secretion, and also suggest an important role for PDE3B in the development of insulin resistance. PDE3B is relatively highly expressed in tissues important for regulation of energy homeostasis, including adipose tissue, pancreas Beta cells, and liver. Although certain aspects of insulin signaling are disrupted in PDE3B KO mice, and these mice exhibited signs of systemic insulin resistance, the KO mice are not diabetic and they accumulated less epididymal fat. We show here that targeted inactivation of PDE3B resulted in increased expression of genes required for fatty acid oxidation and energy dissipation in white adipose tissue (WAT), including UCP1, PGC-1alpha, PPARalpha, enzymes for fatty acid Beta-oxidation, and macromolecules for mitochondrial biosynthesis. Furthermore, AMPK Beta1 subunits, AMPK activity, and phospho-ACC were increased in WAT from PDE3B KO mice. These results provide insight into the significant increases, in PDE3B KO mice, in fatty acid oxidation in isolated epididymal adipocytes, oxygen consumption both in WAT and brown adipose tissue (BAT), and oxygen consumption at whole body level in response to Beta-3 adrenoreceptor agonist stimulation. These data might explain why PDE3B KO mice exhibited phenotypic characteristics of lower epididymal fat and accumulated less body weight under high fat diet treatment. In addition, mRNA levels of macrophage-restricted proteins and enzymes for reactive oxygen species were decreased in epididymal adipose tissue from PDE3B KO mice. Taken together, these results also suggest that in PDE3B KO mice, increased energy dissipation and, perhaps, reduced inflammation in WAT alleviate the insulin-resistance, and prevent frank diabetes in these mice. These results further indicate that in PDE3B KO mice, white epididymal adipose tissue assumes some phenotypic characteristics of BAT, and suggest that PDE3B might serve as an important regulator of lipid and energy metabolism.